This invention relates to the use of recombinant DNA techniques to produce hepatitis B surface antigen (HBsAg).
Hepatitis B virus (HBV) is the infectious agent of serum hepatitis. Infection by this virus is a worldwide health problem; carriers of HBV can suffer from transient or chronic infection, the latter having the potential of progressing to liver cancer. The HBV infectious agent has been identified as the 42 nm Dane particle which contains a lipoprotein coat of hepatitis surface antigen surrounding an internal core particle consisting of a DNA polymerse and the 3200 base pair (bp) DNA genome. HBsAg is found in the serum of HBV carriers mainly in the form of 22 nm spherical particles or filaments and is observed to be the major target for the HBV neutralizing antibody. The 22 nm particles contain two polypeptides of apparent molecular weights of 22,000 and 27,000 daltons. These polypeptides are probably identical, differing only in the presence of glycosylation in the larger peptide. Because of the clinical importance of developing vaccines against HBsAg, a major effort has been undertaken by a number of laboratories to isolate HBsAg protein.
The HBV gene has been cloned into Escherischia coli and the complete nucleotide sequence has been determined, e.g., by Burrel et al. (1979) Nature 279, 43. The HBV DNA is a partially double stranded molecule with a single-stranded gap in one strand (L) and DNA of variable length in the second strand. The HBsAg gene has been cloned in Escherischia coli and has been shown to contain an open reading frame of 680 bp with no intervening sequences. A variety of systems have been employed in order to transfer the HBsAg DNA sequences into a host cell and obtain expression of HBsAg. Varying levels of HBsAg expression have been detected in yeast and mammalian cells transformed with viral vectors such as SV40 and retroviruses. For example, Moriarty et al. 1981 Proc. Natl. Acad. Sci. 78 2606-2610 and Liu et al. 1982 DNA (1) 213-221 describe SV40 vectors that contain the HBsAg gene and are capable of transforming cultured mammalian cells.
Pending U.S. patent application, Hamer et al., Ser. No. 452,783, filed Dec. 23, 1982, entitled "Human Growth Hormone Produced by Recombinant DNA in Mouse Cells", describes a mouse metallothionein (MT)--human growth hormone hybrid gene cloned in a bovine papilloma virus (BPV) vector. Expression of human growth hormone is induced by cadmium or another heavy metal, e.g., zinc. Hamer et al. says, p. 5, that "[t]his vector should be useful for introducing other nonselectable genes into cultured cells, e.g., genes for . . . virus gene products that could be used as vaccines (such as hepatitis B surface antigen)".
Sarver et al (1981) Mol. and Cell. Biol. 1, 486-496; DiMaio (1982) P.N.A.S. USA 79, 4030-4034; and Zinn et al. (1982) P.N.A.S. USA 79, 4897-4901 describe BPV vectors which express, respectively, rat preproinsulin, human beta-globin, and human beta-interferon when used to transform mammalian cells.